The goal of the proposed research is to purify hepatic microsomal hydroxylases and related membrane-bound enzymes and study their interactions in reconstituted enzyme systems, with the intention of establishing the detailed mechanism for the NADH-mediated synergism of NADPH-dependent drug and carcinogen hydroxylations catalyzed by different cytochrome P-450 isozymes. The steady-state and rapid kinetics of cytochrome b5 reduction by NADPH and NADPH-cytochrome P-450 reductase as well as by NADH and NADH-cytochrome b5 reductase will be thoroughly examined by conventional and stopped flow spectrophotometry. The role of mixed function oxidase substrates and of various phospholipids in these reactions will be extensively evaluated. Gel filtration experiments will be performed to ascertain whether cytochrome b5 forms a complex with the NADPH- and NADH-specific flavoprotein, respectively, and to determine the stoichiometry of the components bound in the complex and the magnitude of the binding constants. The effect of NADPH, NADPH-cytochrome P-450 reductase, and cytochrome P-450 on the aerobic steady-state level of NADH-reduced cytochrome b5 in the presence of b5 reductase will be investigated by dual wavelength spectrophotometry. Conversely, we will examine the effect of cytochrome b5 and NADH-cytochrome b5 reductase on the kinetics of reduction and reoxidation of cytochrome P-450, using repetitive scan spectral measurements and stoppped flow spectrophotometry under aerobic or anaerobic conditions as required. In order to establish which isozyme of cytochrome P-450 and what types of mixed function oxidase substrates are subject to the 'synergistic' action of the components of the NADH-linked electron transport chain, we will pursue time course studies on the NADPH-dependent hydroxylation of a variety of substrates of both the phenobarbital-inducible (P-450LM2) and 5,6-benzo-flavone-inducible (P-450LM4) forms of rabbit liver microsomal cytochrome P-450. The influence of cytochrome b5 on the stoichiometry of cytochrome P-450-catalyzed hydroxylation reactions will also be assessed.